1. Field of the Invention
This invention relates generally to immunoadhesins and methods utilizing immunoadhesins. Specifically, this invention relates to methods of producing immunoadhesins, methods of delivering immunoadhesins, methods of treatment using immunoadhesins, and methods of screening immunoadhesins for bioactivity using a replication-deficient adenovirus system.
2. Background Art
Immunoadhesins are antibody-like chimeric fusion proteins comprised of an immunoglobulin gamma heavy chain constant region plus another non-immunoglobulin molecule, such as a cell surface receptor, a cell-adhesion molecule, or a ligand, and are useful for research such as receptor structure-function analysis (Cheon et al., 1994; Heidaran et al., 1995), immunohistochemistry (LaRochelle et al., 1995), ligand identification (Watson et al., 1990), and receptor isolation (Beck et al., 1994) as well as clinical therapeutics (Ashkenazi et al., 1991; Byrn et al., 1990; Chamow et al., 1992; Finck et al., 1994; Jin et al., 1994; Lenschow et al., 1992; Lin et al., 1993; Linsley and Ledbetter, 1993; Wallace et al., 1994; Zheng et al., 1995). Since immunoadhesins can be constructed from a human protein sequence with a desired specificity linked to an appropriate immunoglobulin hinge and constant domain sequence, the binding specificity of interest can be achieved entirely by using human-derived components. Therefore these immunoadhesins are minimally immunogenic to a human subject and safe for therapeutic applications. For example, the immunoadhesin CD4-IgG has been used in human clinical trials. (Hodges et al. "Phase 1 Study of Recombinant Human CD4-Immunoglobulin G Therapy of Patients with AIDS and AIDS-related Complex. Antimicrob. Agents Chemother. 35:2580-6; 1991)). Additionally, since these immunoadhesins can be constructed using various binding or "active" regions, they can be designed for treating specific conditions.
For example, noninfectious ocular inflammation of presumed autoimmune etiology is estimated to cause approximately 10% of severe visual handicap (70,000 cases per year) in the U.S. These diseases cause an inflammation ofthe uveal tract affecting both the anterior and posterior segments of the eye, are chronic degenerative, and potentially blinding, and are collectively known as uveitis. Affected individuals range typically in age from the first to seventh decades, although the second through fourth decades are particularly susceptible. The precise pathogenic mechanisms causing this condition remain elusive. Treatment for these conditions is by necessity nonspecific and entails the use of immunosuppressive agents which provide relief but, unfortunately, can be associated with myriad systemic side adverse effects which can be life threatening. Thus, there clearly is a need for therapeutic alternatives to current treatments that are more specific and avoid such adverse effects. The administration of immunoadhesins described herein represents such a therapeutic alternative.
To date, production of immunoadhesin molecules has entailed the use of transfection of plasmid DNA in a transient fashion or by establishment of stable transfectants (Aruffo et al., 1990; LaRochelle et al., 1995; Linsley et al., 1991). The yields of immunoadhesin obtained have ranged from 0.5-4.5 mg/i following affinity chromatography on immobilized protein A in transient transfection systems (Aruffo et al., 1990; Linsley et al., 1991). Higher concentrations (30-50 mg/l) have been described following the establishment of stable transfectants in large scale production (Haak-Frendscho et al., 1993). While these methods are useful for producing immunoadhesins, they are limited by the inefficiency of transfection and the rather labor intensive nature of establishing stable transfectants (Linsley et al., 1991). This relative lack of efficiency takes on particular significance when screening multiple constructs for activity. It is necessary to produce milligram quantities of recombinant immunoadhesins for preclinical efficacy screening. Ideally, a system for production of an immunoadhesin would combine efficient, high level gene expression, appropriate assembly/post translation modification, and ease of purification.
The methods described herein disclose such a system by providing replication-defective adenovirus vectors comprising nucleic acids encoding immunoadhesins to provide an efficient means of gene transfer into a variety of cell types. Additionally, adenovirus-mediated gene transfer results in high level protein expression. This combination of facile gene transfer and high specific activity provides a rapid means of producing milligram quantities of recombinant molecules.
This inventive system shows marked improvement over other techniques in many respects. Previous techniques involve transient transfection for protein production and result in low transfection efficiencies as well as low amounts of fusion protein production. Cumbersome screening protocols make isolation of protein producing clones time consuming and difficult. In addition, the activity of the chimeric protein can be greatly affected by the production method. The chimera may have different activities based upon whether it is made in bacteria, baculovirus, or mammalian cells.
The present replication-defective adenovirus system involves a method of producing immunoadhesins which is very close to a human system and therefore minimizes undesirable characteristics of a recombinant protein molecule such as incorrect glycosylation and other post-translational protein modifications. Moreover, this system results in transductions at surprisingly much higher efficiency resulting in greater levels of protein production and simplified screening procedures. Thus, the present methods also provide a much-needed, improved, and specific immunotherapeutic method for use in treating uveitis and, in particular, in treating individuals with autoimmune diseases that avoids the adverse side effects of previous treatments.